Despite several decades of studies, the mechanisms that regulate photoreceptor outer segment assembly remain largely unknown. The focus of my laboratory is to understand how the interactions between the retinal pigment epithelium (RPE) and the neural retina regulate outer segment assembly. In our previous studies of the Xenopus laevis embryonic retina, we have shown that removal of the RPE not only disrupts outer segment assembly, but also differentially regulates the protein expression profiles of both photoreceptors and Muller cells. We have also identified factors that, when added to culture, can mimic the presence of the RPE, allowing for normal outer segment assembly and normal retinal protein expression profiles. To facilitate and expedite the delineation of the mechanisms underlying the complex process of photoreceptor outer segment assembly, during the requested grant period, we will incorporate two-dimensional (2-D) separation of proteins followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry into our experimental strategies. We propose to use these powerful proteomic tools to compare the protein expression profiles of the retina, i.e., the retinal proteome, of Xenopus laevis eyes under four well-characterized experimental conditions (differential proteomics): the control whole RPE-neuroretinal proteome; that of RPEdeprived retinas with disorganized outer segments; that of retinas cultured in the presence of a nonmetabolizable glycan that supports normal outer segment assembly ("permissive" glycan) as a positive control; and that of retinas exposed to a "non permissive" glycan, to control for false positives. Inter-gel variability will be minimized and reproducibility enhanced by using 2D Differential In-Gel Electrophoresis (2D-DIGE) and the ETTANtwelve multicasting gel system. Proteins that are up- or downregulated under the different conditions will be identified MALDI-ToF MS, categorized by cluster analysis, and subsequently characterized. We predict that: (1) the majority of the differentially regulated proteins will cluster within three functional groups (cell adhesion molecules, cytoskeletal proteins, and intracellular signaling pathways); and (2) that the majority of the involved proteins will be expressed in photoreceptors and Muller cells. Therefore, we will use these criteria to prioritize our analysis of the differentially regulated proteins. We will then determine if selected target proteins that fulfill these criteria are necessary and sufficient to support outer segment assembly by down regulating their expression using a Morpholino gene knockdown approach followed by quantification of outer segment organization. Our approach will allow us to identify any functional group and/or novel protein that are sufficient for photoreceptor outer segment assembly. These studies will also generate the framework for future project periods in which the precise molecular mechanisms and detailed pathways that control photoreceptor outer segment assembly will be determined. [unreadable] [unreadable]